Controls of corticotrophin-releasing factor output by hypothalamic tissue from fetal sheep in vitro

1989 ◽  
Vol 122 (1) ◽  
pp. 15-22 ◽  
Author(s):  
A. N. Brooks ◽  
L. A. Power ◽  
S. A. Jones ◽  
K. P. Yang ◽  
J. R. G. Challis
Keyword(s):  
G Protein ◽  
Gestational Age ◽  
Potassium Depolarization ◽  
Fetal Sheep ◽  
Corticotrophin Releasing Factor ◽  
Basal Release ◽  
Hypothalamic Tissue ◽  
Negative Feedback Control ◽  
Perifusion System

ABSTRACT Corticotrophin-releasing factor (CRF) is thought to be an important physiological regulator of the pituitary-adrenal axis in fetal sheep and, as such, plays a fundamental role in the initiation of parturition in this species. However, little is known of the controls of CRF secretion from the fetal hypothalamus. We looked for the presence of CRF in fetal hypothalami, and examined whether the hypothalamic CRF concentration or molecular species changed in relation to gestational age. We established an in-vitro perifusion system to examine the release of CRF from perifused hypothalami taken from fetuses at day 100 and day 140 of pregnancy, under basal conditions and in response to potassium depolarization and/or dexamethasone administration. Immunoreactive CRF was present in fetal hypothalami as early as day 100 (2·42 ± 0·99 (s.e.m.) μg/g protein, n = 9) and in similar concentrations at day 140 (2·31 ± 0·69 μg/g protein, n = 9). There was a significant (P < 0·05) increase in hypothalamic CRF content to 14·79 ± 4·09 μg/g protein (n = 16) between day 122 and day 135 of gestation. Using Sephadex G-75 chromatography, hypothalamic extracts at day 100, days 122–135 and day 140 eluted with a single peak of immunoreactivity which corresponded to synthetic ovine CRF(1–41). The basal release of CRF from perifused hypothalami at day 140 (76·6 ± 10·4 pg/fraction, n = 8) was significantly (P < 0·05) greater than at day 100 (50·1 ± 10·2 pg/fraction, n = 11). Dexamethasone significantly inhibited basal CRF release at day 140 of gestation but not at day 100. Potassium depolarization caused a rapid release of CRF in all cases, a response which was independent of gestational age or treatment with dexamethasone. We conclude that the fetal hypothalamus contains immunoreactive CRF as early as day 100 of gestation and that this material may be released when perifused in vitro under basal conditions and in response to a depolarizing agent. The basal release of CRF from perifused hypothalami of day-140 fetuses was greater than at day 100 and was inhibited by dexamethasone, suggesting maturation of negative feedback control of CRF output between days 100 and 140. Since dexamethasone had no effect on potassium-stimulated release of CRF, we suggest that its effects are at sites other than the hypothalamic CRF nerve terminals. Journal of Endocrinology (1989) 122, 15–22

THE PITUITARY-ADRENAL SYSTEM OF THE GENETICALLY OBESE (ob/ob) MOUSE

1975 ◽  
Vol 65 (1) ◽  
pp. 99-107 ◽  
Author(s):  
J. A. EDWARDSON ◽  
C. A. M. HOUGH
Keyword(s):  
Normal Values ◽  
Obese Mice ◽  
Litter Mate ◽  
Corticotrophin Releasing Factor ◽  
Adrenal System ◽  
Pituitary Glands ◽  
Hypothalamic Tissue ◽  
Maintain Body Weight ◽  
Perifusion System

SUMMARY Adult genetically obese (ob/ob) mice which are characterized by adrenal hypertrophy and increased secretion of corticosteroids have considerably increased levels of ACTH in the pituitary gland. At 5 weeks of age there is no difference in the pituitary ACTH content of lean and obese animals and dietary restriction, sufficient to maintain body weight at normal values, reduces the pituitary ACTH content of adult obese mice from 14 times the level found in lean litter-mate controls to almost normal values. Using an in-vitro perifusion system, the release of ACTH from isolated pituitary glands was studied. Pituitaries from lean and obese mice responded similarly to stimulation with a crude extract of hypothalamic tissue containing corticotrophin releasing factor (CRF). The CRF content of the hypothalamus in both groups appears to be similar. In contrast with the high pituitary content, plasma values for ACTH in unstressed obese mice are not increased. The results are discussed in relation to other evidence for a hypothalamic disorder in ob/ob mice.

Effect of hypothalamic neuropeptides on corticotrophin release from quarters of rat anterior pituitary gland in vitro

1984 ◽  
Vol 100 (2) ◽  
pp. 219-226 ◽  
Author(s):  
S. A. Nicholson ◽  
T. E. Adrian ◽  
B. Gillham ◽  
M. T. Jones ◽  
S. R. Bloom
Keyword(s):  
Pituitary Gland ◽  
Anterior Pituitary ◽  
Low Dose ◽  
Physiological Role ◽  
Anterior Pituitary Gland ◽  
High Concentration ◽  
Response Curves ◽  
Corticotrophin Releasing Factor ◽  
Basal Release

ABSTRACT The effect of six hypothalamic peptides on the basal release of ACTH and that induced by arginine vasopressin (AVP) or by ovine corticotrophin releasing factor (oCRF) from fragments of the rat anterior pituitary gland incubated in vitro was investigated. Dose–response curves to AVP and to oCRF were obtained, and the response to a low dose of oCRF was potentiated by a low dose of AVP. Basal release of ACTH was not affected by any of the peptides in concentrations in the range 10−12 to 10−6 mol/l, and only substance P (SP) and somatostatin (SRIF) inhibited significantly the response to oCRF in a dose-related manner. The responses to a range of doses of oCRF or AVP were reduced by 10−8 and 10 − 6 mol SP or SRIF/1, and to a greater extent by the higher dose. Except in the case of 10−6 mol SRIF/1 on the response to AVP, the response was not further diminished by preincubation of the tissue with the peptide before the stimulating agent was added. The inhibition of the responses to AVP or oCRF by 10−9 mol SP/1 was not potentiated by its combination with either 5 × 10−10 or 10−8 mol SRIF/1; the inhibitory effects were merely additive. The results suggest that although SRIF and SP are able to modulate the release of ACTH from the anterior pituitary gland, they do so only at a high concentration. In the case of SRIF these concentrations are several orders of magnitude higher than those reported to be present in the hypophysial portal blood and therefore a physiological role for this peptide in the control of ACTH secretion is unlikely. J. Endocr. (1984) 100, 219–226

Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41

1990 ◽  
Vol 5 (2) ◽  
pp. 159-166 ◽  
Author(s):  
N. G. N. Milton ◽  
E. W. Hillhouse ◽  
S. A. Nicholson ◽  
C. H. Self ◽  
A. M. McGregor
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Corticotrophin Releasing Factor ◽  
Tissue Extracts ◽  
Rat Pituitary ◽  
Hypothalamic Tissue ◽  
Dose Dependent

ABSTRACT Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

Somatostatin inhibits VIP-stimulated amylase release from perifused guinea pig pancreatic acini

1988 ◽  
Vol 254 (2) ◽  
pp. G217-G223 ◽  
Author(s):  
P. Singh ◽  
I. Asada ◽  
A. Owlia ◽  
T. J. Collins ◽  
J. C. Thompson
Keyword(s):  
Guinea Pig ◽  
Fold Increase ◽  
Optimal Conditions ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
In Vitro System ◽  
Basal Release ◽  
Perifusion System ◽  
Chamber Size

We have examined the direct effect of somatostatin (SRIF) on basal and stimulated amylase release from guinea pig pancreatic acini using the in vitro method of continuous perifusion. The optimal conditions of flow rate, chamber size, acinar cell volume per chamber, and period of secretagogue infusion were defined for the perifusion system. The kinetic profile of amylase release in response to cholecystokinin-octapeptide (CCK-8), vasoactive intestinal peptide (VIP), and SRIF was studied. Under optimal conditions, the acini were found to remain equally responsive to an ED50 dose of CCK-8 (0.5-0.8 nM) for 12 h of perifusion. The duration of amylase response to any given dose of CCK-8, given for the optimal period of 5 min, was 80-100 min. The total amylase released minus the basal release divided by 90 min (delta response) in response to the maximum effective (Maxeff) dose of CCK-8 (100 nM) was 14,667 +/- 1,433 U/l (amounting to a 10-fold increase compared with basal values). When compared with the amount of total delta amylase released in response to the Maxeff dose of CCK, the total amylase released in response to the Maxeff doses of SRIF (1 microM) and VIP (10 nM) was 10-21% and 51-59%, respectively. SRIF (100 nM) significantly decreased VIP- (0.1-1.0 nM) stimulated amylase release by 45-70% in the perifusion method of study but had no significant effect on the CCK-stimulated amylase release. This suggests that the perifusion method can be used for investigating the mechanism of SRIF-mediated inhibition of VIP effects on amylase release in an in vitro system.

Corticotrophin-releasing factor-like immunoreactivity and bioactivity of human fetal and adult hypothalami

1986 ◽  
Vol 108 (2) ◽  
pp. 171-180 ◽  
Author(s):  
J. F. Ackland ◽  
S. J. Ratter ◽  
G. L. Bourne ◽  
L. H. Rees
Keyword(s):  
Molecular Weight ◽  
Gestational Age ◽  
Anterior Pituitary ◽  
Pituitary Cell ◽  
Perfusion System ◽  
Corticotrophin Releasing Factor ◽  
Tissue Extracts ◽  
Pituitary Glands ◽  
Hypothalamic Tissue ◽  
Molecular Weight Form

ABSTRACT Corticotrophin releasing factor-like immunoreactivity (CRF-LI) and bioactivity, and arginine vasopressinlike immunoreactivity (AVP-LI) have been measured in extracts of human fetal and adult hypothalamic tissue and their development with the gestational age of the fetuses (12–27 weeks) studied. CRF-LI was measured by a radioimmunoassay developed for ovine corticotrophin-releasing factor (oCRF-41). Corticotrophin-releasing factor bioactivity was measured in a rat isolated anterior pituitary cell perfusion system. CRF-LI and bioactivity and AVP-LI were all detectable in fetal hypothalamic extracts from 12 to 13 weeks of gestational age. CRF-LI was also present in human fetal pituitary glands from 12 weeks of gestational age. The concentration of CRF-LI in the fetal hypothalamic extracts (9·2±11·4 ng/g, mean ± s.e.m., n = 33) showed no significant correlation with the gestational age of the fetuses. However the concentration of AVP-LI (25·0–36·8 ng/g, n = 17) did show a positive correlation (r = 0·508, P<0·05) with gestational age, as did the concentration of CRF bioactivity (471·3–556·3 ng ACTH released/g tissue, n = 13, r = 0·725, P < 0·01). The CRF bioactivity of all fetal hypothalamic extracts was potentiated by the addition of synthetic human (h)AVP, but the bioactivity of the adult hypothalamic extracts was not, presumably because of the higher levels of AVP-LI already present in the adult extracts. Pretreatment of tissue extracts with antisera to oCRF-41 and/or hAVP reduced the CRF bioactivity of all hypothalamic extracts. Sephadex chromatography of fractions which co-eluted with synthetic oCRF-41 or hAVP contained CRF bioactivity and this bioactivity was potentiated when synthetic hAVP or oCRF-41, respectively, were added to the fractions. However, a larger molecular weight form of CRF-LI (8000–10 000 daltons), which was observed only in fetuses of 20 weeks of gestational age or less, did not contain any significant CRF bioactivity. J. Endocr. (1986) 108, 171–180

Dexamethasone inhibits ovine corticotrophin-releasing factor (oCRF), arginine vasopressin (AVP), and oCRF + AVP stimulated release of ACTH during the last third of pregnancy in the sheep fetus

1987 ◽  
Vol 65 (6) ◽  
pp. 1186-1192 ◽  
Author(s):  
Laurie J. Norman ◽  
John R. G. Challis
Keyword(s):  
Negative Feedback ◽  
Arginine Vasopressin ◽  
Late Pregnancy ◽  
Feedback Effect ◽  
Plasma Acth ◽  
Fetal Sheep ◽  
Corticotrophin Releasing Factor ◽  
Basal Release ◽  
Sheep Fetus ◽  
Acth Release

We examined the hypothesis that in fetal sheep during late pregnancy exogenous glucocorticoids might affect differentially the pituitary response, measured as changes in plasma ACTH concentrations, to the systemic administration of ovine corticotrophin-releasing factor (oCRF), arginine vasopressin (AVP), or oCRF + AVP. At d 113–116 of pregnancy, equimolar injections of oCRF and AVP given separately provoked similar significant increases in plasma ACTH; the change in ACTH over basal values was significantly greater than the sum of the two separate responses when AVP + oCRF were given together. Exogenous dexamethasone did not affect basal ACTH concentrations, but suppressed significantly the responses to oCRF, AVP, and oCRF + AVP. At d 126–130, there was a significant ACTH response to CRF alone and to AVP + oCRF, but not to AVP alone. The response during the first 30 min postinjection to oCRF was significantly less than that to AVP + oCRF. Plasma Cortisol rose after each peptide injection. Exogenous dexamethasone suppressed both basal and stimulated responses to each peptide. At the amounts injected, there was no significant ACTH or Cortisol response to oCRF, AVP, or oCRF + AVP at d 136–140, but dexamethasone suppressed basal ACTH and Cortisol concentrations at this time. We conclude that stimulated, but not basal, release of ACTH is subject to the negative feedback effect of exogenous glucocorticoid by d 113–116 of gestation in fetal sheep. Both basal and stimulated release of ACTH and Cortisol are suppressed after d 125. At the amount of exogenous dexamethasone given, oCRF, AVP, and oCRF + AVP-stimulated responses are affected similarly. Our results suggest different controls of basal and stimulated ACTH release from the pituitary at d 113–116 of gestation. Our findings would be consistent with the pituitary as a level of action for the negative feedback effect of corticosteroids on stimulated ACTH release throughout the last third of pregnancy in fetal sheep.

Modulation by endogenous opioid peptides of the secretion of LHRH from cockerel (Gallus domesticus) mediobasal hypothalamic tissue

1987 ◽  
Vol 114 (1) ◽  
pp. 103-110 ◽  
Author(s):  
S. C. Stansfield ◽  
F. J. Cunningham
Keyword(s):  
Opioid Peptides ◽  
Inhibitory Influence ◽  
Mediobasal Hypothalamus ◽  
Endogenous Opioid Peptides ◽  
Endogenous Opioid ◽  
Superfusion System ◽  
In Vitro Superfusion ◽  
Basal Release ◽  
Hypothalamic Tissue

ABSTRACT An in-vitro superfusion system was used to study the effects of the endogenous opioid peptides [Met]-enkephalin (and its long-lasting analogue [d-Ala2, Met]-enkephalinamide), [Leu]-enkephalin and β-endorphin and of the opiate antagonist naloxone, on the secretion of LHRH from the mediobasal hypothalamus of the cockerel. The effects of the compounds on both basal release of LHRH and on release stimulated by a depolarizing pulse of increased extra-cellular potassium ion (64 mmol/l) were investigated. None of the endogenous opioid peptides altered basal release of LHRH; however, both [Met]-enkephalin (10 μmol/l) and [d-Ala2,Met]-enkephalinamide (1 μmol/l) significantly (P<0·05) reduced the response to depolarization. Neither [Leu]-enkephalin nor β-endorphin (0·1–10 μmol/l) were effective. Naloxone (1 μmol/l) administered alone significantly (P<0·05) increased basal release of LHRH and abolished the inhibitory effects of [Met]-enkephalin and [d-Ala2,Met]-enkephalinamide on depolarization-induced release. These results suggest that the endogenous opioid peptides exert a tonic inhibitory influence on LHRH secretion by the mediobasal hypothalamus of the cockerel. J. Endocr. (1987) 114, 103–110

Changes in pituitary responses to synthetic ovine corticotrophin releasing factor in fetal sheep

1985 ◽  
Vol 63 (11) ◽  
pp. 1398-1403 ◽  
Author(s):  
Laurie J. Norman ◽  
Stephen J. Lye ◽  
Mary E. Wlodek ◽  
J. R. G. Challis
Keyword(s):  
Plasma Cortisol ◽  
Late Pregnancy ◽  
Sampling Interval ◽  
Plasma Acth ◽  
Fetal Sheep ◽  
Corticotrophin Releasing Factor ◽  
Spontaneous Labour ◽  
Negative Feedback Control ◽  
Relationship Of ◽  
The Relationship

The rise in cortisol in fetal sheep during late pregnancy has been related to increased responsiveness of the adrenal to ACTH. Most reports have suggested that plasma ACTH concentrations rise coincident with or after the prepartum increase in cortisol. To reexamine the relationship of cortisol with basal immunoreactive ACTH (IR-ACTH) throughout the last 40 days of pregnancy and to determine changes in fetal pituitary responsiveness during this time, we measured basal and synthetic ovine corticotrophin-releasing factor (oCRF) (10 ng – 10 μg) induced rises in ACTH and cortisol in fetal sheep at days 110–115, 125–130, and 135–140 of pregnancy. The fetuses were catheterized on day 105–120 and entered spontaneous labour at > 140 days. Basal IR-ACTH (picograms per millilitre ± SEM) rose from 16.7 ± 2.9 pg/mL at day 110–115 to 34.8 ± 8.7 pg/mL at day 141–145. There was a significant effect of time on basal ACTH concentrations with a mean increase of approximately 5 pg ACTH per millilitre of plasma per 5-day sampling interval. Plasma cortisol changed gradually between day 110 and 125 of gestation and then more rapidly to term. At day 110–115 of gestation there was no significant change in plasma ACTH after 10 or 100 ng oCRF, but there was a significant increase in ACTH after 1 μg of oCRF. Plasma cortisol did not change after any CRF injection. The change in IR-ACTH after oCRF at day 125–130 of gestation was significantly greater than that at day 110–115. Plasma cortisol concentrations were elevated following 1- and 10-μg injections of oCRF. At day 135–140, significant rises in plasma ACTH were seen in response to 1 and 10 μg oCRF, but the response was less than that at day 125–130. In contrast, the response of plasma cortisol was significantly greater than at any of the other times in gestation. We conclude the following: (i) basal ACTH concentrations rise before the major prepartum increase in plasma cortisol; (ii) pituitary responsiveness to oCRF, measured as ACTH in plasma, increases between days 110–115 and 125–130 of gestation. The ACTH response decreased at day 135–140, perhaps reflecting negative feedback control by the rising basalcortisol concentrations; (iii) adrenal responsiveness increases progressively between days 110–115 and 135–140 of gestation, as reflected by changes in the plasmacortisol concentration in response to endogenously released ACTH.

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